Construction Of The Transgenic Plant And Preliminary Functional Studies Of BnRGS1and BnGA1in Brassica Napus

G protein mediated signal transduction pathways in eukaryotes is one of the most important and conservative signaling pathways, which responsible for recognition, perception and transduction external stimuli. Heterotrimeric G protein is consist of three subunits. including Ga subunit、Gβsubunit and Gy subunit. The regulator of G protein signal transduction (RGS) is directly combined with activated Ga subunit, which accelerating the hydrolysis of GTP to GDP, thus terminate G protein signal transduction pathways.We have successfully cloned BnRGS1gene by using the Brassica rape gene bank in previous work. In order to further study on BnRGS1and BnGAl gene function in Brassica napus, we constructed the transgenic plant of BnRGS1and BnGA1through tissue culture technology of Brassica napus, and investigated the function of the two genes in the presents study. The main results are as follows:(1) According to the information of Genebank, using ’yangyou6’ as the material, we cloned BnRGS1and BnGAl gene sequences.(2) The BnRGS1-GFP recombination vector was transferred into callus cells by agrobacterium GV3101. BnRGSl was localized on the membrane through fluorescent observation of the positive callus carrying CaMV35S::BnRGSl.(3) The gene expression of restructuring CaMV35S::BnRGS1and CaMV35S::BnGA1interference and overexpression positive callus was analyzed. The expression of rab18and RD29was high in RGS overexpression callus than in wild type with the ABA treatment, and the difference reduced with the increase of ABA concentration between RGSo and wild type. The expression of rab18/RD29in GAi was higher than that in WT when treated with ABA in4h. The results indicated that BnRGS1and BnGA1were involved in ABA signaling in Brassica napus. The ABA content was increased after the treatment of6%glucose, while the expression of ABA synthetase genes (ABA1、ABA2and NCED3) was increased and the expression of ABA degradation enzyme gene (cyp707A1) was decreased with the treatment of6%glucose. The difference of ABA synthetase/degradation genes (ABA1、ABA2and cyp707A1) was not significant among RGSo, RGSi. GAi and wild type with the treatment of3%glucose. These indicated that BnRGS1was involved in the regulation of ABA synthesis related to glucose. (4) With hypocotyle as explants, effects of various factors on regenerative seedling were studied in tissue culture of Brassica napus, and the effective hypocotyle regenerative system was established. We developed and identified two BnRGSl interference plants and one BnGAl interference plant successfully.(5) CaMV35S::BnRGSl expression vector was transferred into Arabidopsis mutant rgs1-2and two positive complementary transgenic plants were obtained by antibiotics screening.